In vitro formation of axillary buds by immature shoots of Ponderosa pine

Transcription

1 Plant Cell, Tissue and Organ Culture 26: , Kluwer Academic Publishers. Printed in the Netherlands. In vitro formation of axillary buds by immature shoots of Ponderosa pine Yiqun Lin j, Michael R. Wagner I & L.J. Heidmann 2 1School of Forestry, Box 4098, Northern Arizona University, Flagstaff, AZ USA; 2USDA Forest Service, Rocky Mountain Forest and Range Experiment Station, 700 S. Knoles Drive, Flagstaff, AZ 86001, USA Received 22 August 1990; accepted in revised form 12 April 199t Key words: Immature shoot explants, in vitro culture, mature ponderosa pine, Pinus ponderosa Dougl ex Laws Abstract Axillary buds were induced from immature shoot explants taken from terminal buds of branches from 29- and 34-year old ponderosa pines (Pinus ponderosa Dougl ex Laws). The effect of collection time, position on the donor tree from which the explants were taken, and plant growth regulators on axillary bud formation was investigated. Explants from branches taken in late October formed axillary buds, whereas explants from branches collected in February 1988 produced a large amount of callus. The ability to form axillary buds was significantly greater for explants from the upper crown than from the lower portion of the tree. Explant elongation occurred and basal needle primordia swelled on Murashige & Skoog media (MS) containing 2.2/zM 6-benzyladenine (BA) and 5.4/zM naphthalenacetic acid. When transferred to a MS medium containing 4.4 ~M BA, 59% of explants formed axillary buds. Introduction Generally, in vitro propagation of mature trees is preferable over that of seedlings or embryos because some important traits may not be expressed until the trees reach maturity (Bonga 1981, 1987). However, most species have only been vegetatively propagated from seedling tissue or embryos (Bonga 1981, 1987). Therefore, it is desirable to develop techniques for vegetative propagation of mature trees. There are several methods to achieve regeneration by in vitro propagation, such as callus culture, suspension culture, and axillary bud induction (John 1983). In this context, axillary bud induction is the formation of buds from axillary meristems produced by immature shoots. In vitro induction of axillary buds capable of forming shoots is a widely used method for the propagation of flowering plants and trees. Clones, derived from axillary buds, maintain genetic stability with less risk of mutation than may arise during organogenesis from callus or suspension culture (Pierik 1987; Vasil & Vasil 1980). Axillary bud induction by tissues from mature conifers has been reported for only a few species. Horgan (1987) reported that axillary shoots were produced from explants of mature Pinus radiata (D. Don). Plantlets were then developed from rooted axillary shoots. Gupta & Durzan (1985) obtained axillary buds from shoots collected from mature Douglas-fir (Pseudotsuga menziesii Franco.) and sugar pine (Pinus lambertiana Dougl). Bonga (1977) obtained lateral buds from year old balsam fir [Abies balsamea (L) Mill]. Ball (1987) reported the induction of axillary buds from stem explants of mature Sequoia sernpervirens [(D Don) Endl]. However, no reports document the in vitro induction of axillary buds from mature ponderosa pine.

2 162 The aim of this study was to develop a method for inducing axillary buds from explants of immature shoots of mature ponderosa pine. Three factors were considered: - collection date, -position on the tree from where the explants were taken, and - growth regulators. Materials and methods Plant materials Two ponderosa pines were chosen randomly from 20 trees growing in a study plot south of Parks, Arizona, in the Kaibab National Forest. The two trees were 29 and 34 years old. From each tree, 20 branches from the top and 20 branches from the lower crown were collected. The branches were stored in plastic bags containing wet paper towels in a refrigerator (2-4 C) for 2 days. Branches were collected on February 5, 1988 and October 28, Preparation of buds and media Terminal buds (20 from the top and 20 from the bottom of the tree) with I cm of subtending stem tissue were washed with 70% ethanol to remove surface resin from the buds. The buds were disinfested using a five-step procedure including: soaking for 2 min in 1% Alconox, 1 soaking in 0.6% sodium hypochlorite for 15min, soaking in 5% H20 2 for 15 min, soaking in 2% of 50% Benlate for 2min, and finally rinsing with sterilized distilled water several times until water appeared clean. Difco Bacto-Agar media were sterilized in an 2 2 o autoclave at 1.4 kg/cm pressure and 121 C for 20min. The media were adjusted to ph 5.8 before sterilization. Culturing procedures The disinfested immature shoots were dissected from subtending stem tissue. The shoot apex was removed and discarded and each immature stem was cut into 3 transverse segments, each about 3-4 mm long. Scales were removed from each segment and the explants were placed on Murashige & Skoog (1962) medium (MS) containing 2.2/zM 6-benzyladenine (BA) and 5.4/xM naphthalenacetic acid (NAA). Each petri dish sealed with Parafilm contained 3 explants from each immature shoot. The concentrations of Difco Bacto-agar and sucrose were 0.8% and 3%. Cultures were incubated for 2 weeks in the initiation passage. They were then transferred onto the same medium for a second culture passage of 4 weeks. In the second and the third culture, explants were placed individually in 50 ml Erlenmeyer glass flasks (1 explant/ flask) covered with rubber plugs. The third culture was designed to test the effect of growth regulators on axillary bud formation. The explants were transferred onto MS media containing one of the following four combinations of growth regulators: - 22/zM BA + 5.4/xM NAA, -22/zM BA, - 4.4/xM BA + 2.7/zM NAA, and -4.4/xM BA. Explants were maintained on the four media for 4 weeks. Axillary bud formation was determined at the end of the third culture period. The frequency of axillary bud formation was calculated by dividing the number of budded explants by the number of cultured explants. Environmental conditions Explants were grown in plastic Petri dishes (5.5 cm 1.5 cm) in the first culture and maintained in glass flasks in the rest of the cultures. Explants were maintained in a growth chamber with a temperature of I C, photoperiod of 1. Trade names are used for brevity and specificity and do not imply endorsement by USDA or NAU to the exclusion of equally suitable products.

3 163 16"8 light/dark, and fluence rate of 82 ~ mol m sec from cool white fluorescent tubes. Statistical analysis Two trees represent 2 independent replications. Since 20 immature shoots were cut into 3 pieces, the total number of explants were 60 from top and 60 from the bottom of each tree. The data for Chi-square analysis were based on recording the number of budded explants from the top and the bottom of the tree and from each growth regulator treatment at the end of the third culture. Chi-square analysis (Zar 1984) was applied to test the effect of the position on the donor tree from which the explants were taken and the four growth regulator treatments on axillary bud formation. Two steps were used in each test. - Chi-square analysis was conducted on individual replicates followed by a test for heterogeneity. -When heterogeneity tests showed that the two replicates were homogeneous, data from the replicates were combined to test the effect of the treatment by Chi-square analysis (Zar 1984). The subdividing contingency table procedure of Chi-square was used to determine the optimum growth regulator treatment (Zar 1984). Results and discussion Description of axillary bud formation The explants collected in October began to elongate along the stem axis after 10 days in culture. They grew rapidly and reached 10 to 15 mm within 5 days. One week after transfer into the second culture medium, the explant elongation stopped. Subsequently, the basal tissue and basal needle primordia of 75% of the explants swelled. In the third culture medium, some swelled needle primordia gave rise to scale-enclosed and well-formed axillary buds, which had a vascular connection with the main axis of the immature shoot as shown in histological examination. For each explant that formed axillary buds, the number of axillary buds ranged from 2-4. Collection date effect Morphogenesis of cultured immature shoot segments was greatly influenced by the date of collection. Explants from branches collected in February started forming white and loose calluses on the tenth day after initiation. None of the explants elongated or formed axillary buds. In contrast to the winter buds, explants from branches collected in October elongated, basal tissue and needle primordia swelled, and axillary buds gradually formed from the swollen needle primordia. Collecting time is often considered a critical factor for successful vegetative propagation (Bonga 1987). Adventitious shoots of Pinus mugo Turra, Picea abies [(L) Karst], and Larix decidua Mill were induced more easily from tissues collected in spring and autumn than during other seasons (Bonga 1984; Bonga & Von Aderkas 1988). Selby & Harvey (1985) reported a strong effect of time of year on the ability of Sitka spruce [Picea sitchensis (Bong.) Carr.] explants to form adventitious buds. When collections were made from December to April, the best results were achieved in March prior to natural bud flush. Explant viability and callus growth of Scots pine (Pinus sylvestris L) was found to be the greatest when explants were taken in December, January, and from April to July (Hohtola 1988). Ball et al. (1978) reported that shoots of Sequoia sempervirens (L Mill) collected in autumn took less time to form buds than the shoots collected in summer. However, Franclet et al. (1987) reported that the success of in vitro culture of mature Sequoia was unaffected by the date of excision. Time effect on explant behavior in vitro has been considered to result from the physiological status of the donor plant and hormone levels at the time of excision (Hohtola 1988; Selby & Harvey 1985). In our study, axillary buds formed only on the material collected in October. This result is correlated with the formation of lateral meristems at that time (Bonga 1987; Kozlowski 1971a).

4 164 Position effect The explants from the upper crown began to elongate earlier and more rapidly than the ones from the lower crown. The explants from the top of the three became swollen about 3 days earlier than ones from the lower crown of the same tree. After the explants stopped forming buds in the fourth week on the third culture medium, the frequency of bud formation was measured. The frequency of axillary bud formation was higher in explants collected from the top of the trees than from those collected from the lower crown. In one replicate the frequency of axillary bud formation by explants from the top and from the bottom crowns were 53% and 18%, respectively. Another replication gave a frequency of 55% for explants on the top of the trees and 41% for those from the lower crown. When data of these two replicates were combined, a statistically significant difference (X 2 = 9.06, p < 0.05) resulted in the frequency of axillary bud formation between top and lower crown explants of the trees. Success of vegetative propagation is highly associated with positions on the tree from which the cutting is taken (Evers 1987; Kozlowski 1971b; Roulund 1973). Roulund (1973) demonstrated that the ability to root increased gradually as cuttings were taken from the top to the bottom of Picea abies. Douglas-fir ( Pseudotsuga menziesii) shoots derived from buds collected from lower branches rooted better than those from upper branches (Evers 1987). However, the importance of positional effect on axillary bud formation in vitro has not been reported previously. Wareing (1957) and Moorby & Wareing (1963) studied growth of branches of Pinus sylvestris and Larix leptolepis as related to their positions on the tree. They showed that branches at the top of the tree grew more vigorously than those at the bottom of the tree. The amount of growth and the growth rate of branches decreased gradually from the top to the bottom. The reduction of growth by the lower branches was referred to as ageing (Wareing 1957). Similar results were obtained by Forward & Nolan (1964) in Pinus resinosa. Ageing occurs earlier in the bottom of a tree and gradually develops toward the top of the tree (Moorby & Wareing 1963). Position also influenced callus proliferation of Scots pine explants. Hohtola (1988) reported that the amount of callus proliferation increased on explants from the bottom to the top of the crown. In our study, the fact of higher frequency of bud formation from the top crown than from the bottom crown is likely due to the ageing effect. Growth regulator effect Comparing the individual treatments, Chi-square analysis suggested that the medium with 4.4/xM BA is the most effective treatment (X 2= 10.5, p < 0.05), resulting in 59% of the explants forming axillary buds. Two interesting observations were made concerning growth regulator treatments. First, increasing the concentration of BA from 4.4 to 22/xM did not further stimulate axillary bud formation (Fig. 1). Campbell & Durzan (1975) found that a high concentration of BA inhibited adventitious bud development from hypocotyl tissue of Picea glauca. Von Arnold & Eriksson (1979) noticed that a cytokinin concentration higher than 5 x 10-5 M stimulated callus formation on the swollen needle primordia of Norway spruce (Picea abies (L) Karst). Secondly, within the two pairs of treatments (A-B and C-D) having the same amount of BA, the frequency of axillary bud formation was lower when NAA was present (treatments A and C) than when it was absent (treatments B and D) (Fig. 1). The opposite was true for percent callus formation. Treatments having both BA and NAA (A and C) had higher percent callus I: O i. O M o 2o 0 A B C Growth Regulator Treatment //J 7/, Fig. 1. Effect of growth regulators on axillary bud (hatched bars) and callus (open bars) formation assessed at the end of the third culture passage. The growth regulator treatments used were, (A) = 22/zM BA + 5.4/xM NAA, (B) = 22/xM BA, (C) = 4.4/.~M BA + 2.7/xM NAA, and (D) = 4.4/xM BA. // // i/j D

5 165 formation than treatments with only BA (B and D) (Fig. 1). Callus formed both before and after axillary bud formation. When callusing occurred before the time of axillary bud formation, explants never formed axillary buds. These facts suggest that NAA stimulated callus formation and inhibited axillary bud formation. Usually, auxin is required to induce callus; cytokinin stimulates shoot formation and shoot development. Rancillac (1977, 198l) found that auxin did not stimulate axillary bud development but stimulated callus formation. Bornman (1984) also reported that the development of axillary buds was obtained without callus formation when BA was applied alone. BA alone was used to stimulate needle primordia growth and to produce new shoots from fascicle primordia of mature radiata pine (Horgan 1987). Acknowledgements We thank the USDA Forest Service, Rocky Mountain Forest and Range Experiment Station for allowing us to use their research facilities. Dr Richard W. Tinus reviewed the manuscript. Larry Sandoval provided general laboratory assistance. This research was supported by Mclntire Stennis research funds and the Northern Arizona University Organized Research Program. References Ball EA (1987) Tissue culture multiplication of Sequoia. In: Bonga JM & Durzan DJ (Eds) Cell and Tissue Culture in Forestry, Vol 3 (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster Ball EA, Morris DM & Rydelius JA (1978) Cloning of Sequoia sempervirens from mature trees through tissue culture. Proc Round Table Conf. 'In vitro' Multiplication Woody Species (pp ). Gembloux, Belgium, June Bonga JM (1977) Organogenesis in in vitro cultures of embryonic shoots of Abies balsamea (balsam fir). In Vitro 13:41-48 Bonga JM (1981) Organogenesis in vitro of tissues from mature conifers. In Vitro 17: Bonga JM (1984) Adventitious shoot and root formation in tissue cultures of mature Larix decidua. In: Hanover J, Karnosky D & Keathley D (Eds) International Symposium of Recent Advances in Forest Biotechnology (pp 64-68). Michigan Biotechnology Institute, Traverse City, Michigan Bonga JM (1987) Clonal propagation of mature trees: problems and possible solutions. In: Bonga JM & Durzan DJ (Eds) Cell and Tissue Culture in Forestry, Vol 1 (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster Bonga JM & Von Aderkas P (1988) Attempts to micropropagate mature Larix decidua Mill. In: Ahuja MR (Ed) Somatic Cell Genetics of Woody Plants (pp ). Kluwer Academic Publishers, Dordrecht, Boston, London Bornman CH (1984) Application of in vitro culture technology in clonal forestry. In: Hanover J, Karnosky D & Keathley D (Eds) International Symposium of Recent Advances in Forest Biotechnology (pp ). Michigan Biotechnology Institute, Traverse City, Michigan Campbell RA & Durzan DJ (1975) Induction of multiple buds and needles in tissue cultures of Picea glauca. Can. J. Bot. 53: Evers PW (1987) Correlations within the tree. In: Bonga JM & Durzan DJ (Eds) Cell and Tissue Culture in Forestry. Vol 2 (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster Forward DF & Nolan NJ (1964) Growth and morphogenesis in the Canadian forest species. Can. J. Bot. 42: Franclet A, Boulay M, Bekkaoui F, Fouret Y, Verschoore- Martouzet B & Walker N (1987) Rejuvenation. In: Bonga JM & Durzan DJ (Eds) Cell and Tissue Culture in Forestry, Vol 1 (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster Gupta PK & Durzan DJ (1985) Shoot multiplication from mature trees of Douglas-fir (Pseudotsuga menziesii) and sugar pine (Pinus lambertiana). Plant Cell Rep. 4: Hohtola A (1988) Seasonal changes in explant viability and contamination of tissue cultures from mature Scots pine. Plant Cell Tiss. Org. Cult. 15: Horgan K (1987) Pinus radiata. In: Bonga JM & Durzan DJ (Eds) Cell and Tissue Culture in Forestry, Vol 3 (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster John A (1983) Tissue culture of coniferous trees. In: Dodds JH (Ed) Tissue Culture of Trees (pp 6-21). AV1 Publishing Co, Inc, Westport, Connecticut Kozlowski TT (1971a) Bud development and shoot expansion. In: Kozlowski TT (Ed) Growth and Development of Trees, Vol 1 (pp ). Academic Press, New York, London Kozlowski TT (1971b) Maturation or phase change. In: Kozlowski TT (Ed) Growth and Development of Trees, Vol 1 (pp ). Academic Press, New York, London Moorby J & Wareing PF (1963) Ageing in woody plants. Ann. Bot. 27:291-31)9 Murashige T & Skoog F (1962) A revised medium for rapid growth and bio-assays with tobacco tissue cultures. Physiol. Plant. 15: Pierik RLM (1987) Vegetative propagation. In: Pierik RLM (Ed) In vitro Culture of Higher Plants (pp ). Martinus Nijhoff Publishers, Dordrecht, Boston, Lancaster Rancillac M (1977) Mise au point d'une m6thode de multiplication v6gdtative "in vitro' du pin maritime (Pinus pinaster

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